The Influences About Macrophages Release Of TNF-α And The Mechanism Challenged With Novel HIV-derived MicroRNA99

Purpose:To investigate the influences about macrophages release of TNF-α and the mechanism challenged with novel HIV-derived micro RNA99.Methods:(1)The construction of macrophages model With phorbol myristic acid(PMA)stimulates the human monocytes leukemia cells differentiate into macrophages,observing the morphology of them in inverted microscope.(2)The influences about macrophages release of TNF-α challenged with novel HIV-derived micro RNA99 Chemical synthesis materials were used to infect macrophages and the research groups were as following:Experimental group :macrophages + transfection reagent + micro RNA99 Control group 1 :macrophages + transfection reagent + micro RNATAR Control group 2 : macrophages + Lipid A Control group 3 : macrophages + medium Incubated in incubator at 37℃and 5% CO2 atmosphere,then collect supernatant after12 hours.(3)The mechanism of HIV-derived micro RNA99 stimulates the release of TNF-α in macrophagesⅰTLR8 plasmids transfection and experimental group as follows Experimental group a:macrophages + nucleartransfection solution+TLR8 plasmids Control group a :macrophages + nucleartransfection solution+plasmids without TLR8 Incubated in incubator at 37 ℃ and 5% CO2 atmosphere,Viewing the cells fluorescence under the confocal fluorescence microscope and collect cells after 48 hours.ⅱMicro RNA99 and Lipid A were used to infect macrophages and the research groups were as following:Experimental group a : macrophages-TLR8 plasmids + micro RNA99+ transfection reagent Control group a:macrophages-plasmids without TLR8 + micro RNA99+ transfection reagent Experimental group b:macrophages-TLR8 plasmids + Lipid A Control group b:macrophages-plasmids without TLR8 + Lipid A Incubated in incubator at 37℃and 5% CO2 atmosphere,then collect supernatant after24 hours.(4)Detection methods(1)Observing the morphology of THP-1 in inverted microscope.(2)Viewing the cells fluorescence under the confocal fluorescence microscope to determining the rate of plasmids transfection.(3)Western Blot was applied to examine TLR8 expression in macrophages of each group.(4)The concentrations of TNF-α in supernatant were determined by ELISA.Results:(1)THP-1 differentiate to macrophages observation THP-1 in a round or class round,a single suspension or grape bunches aggregating state differentiate into a fusiform or polygon adherent cells,namely macrophages.(2)The influences about macrophages release of TNF-α challenged with novel HIV-derived micro RNA99 In the experimental group(add in micro RNA99)and control group 1(add in micro RNATAR),the level of TNF-α in supernatant statistically significant higher than the control group 2(add in Lipid A)and control group 3(add in medium)(p<0.05);there is no statistically difference between the control group 2 and control group 3(p>0.05);However,the level of TNF-α in supernatant of experimental group is statistically significant higher than the control group 2(p<0.05).(3)The mechanism of HIV-derived micro RNA99 stimulates the release of TNF-α in macrophagesⅰViewing the cells fluorescence under the confocal fluorescence microscope to determining the rate of plasmids transfection.The phenomenon that there are green fluorescence in macrophages means the process of transfection of TLR8-plasmids and plasmids without TLR8 is successfulⅱ Western Blot was applied to examine TLR8 expression in macrophages of each group.The level of TLR8 protein expression in TLR8-plasmids macrophages is statistically significant lower than plasmids without TLR8(p<0.05).ⅲ The concentrations of TNF-α in supernatant were determined by ELISA.In the experimental group a,the level of TNF-α in supernatant statistically significant lower than the control group a(p<0.05);there is no statistically difference between the experimental group b and control group b(p>0.05).Conclusion:1.HIV-derived micro RNA99 promotes the release of TNF-α in macrophages.2.HIV-derived micro RNA99 impacts macrophages release TNF-α in a TLR8-dependent way.